Vol.:(0123456789)1 3

Forensic Toxicology (2020) 38:95–107 https://doi.org/10.1007/s11419-019-00491-0

ORIGINAL ARTICLE

Evidence of enzyme‑mediated transesterification of synthetic cannabinoids with ethanol: potential toxicological impact

Orapan Apirakkan1 · Ivana Gavrilović1,2 · Giuseppe Floresta1,3 · Cheyanne Pierre1,2 · Annelies Cannaert4 · Christophe P. Stove4 · Paul I. Dargan5,6 · David A. Cowan1 · Lewis Couchman7 · Vincenzo Abbate1

Received: 21 May 2019 / Accepted: 2 July 2019 / Published online: 13 July 2019 © The Author(s) 2019

AbstractPurpose Synthetic cannabinoids (SCs) represent a large proportion of novel psychoactive substances on the black market and have caused a number of deaths. Polydrug use including combination of SCs and ethanol could further complicate the toxicological impact. To the best of our knowledge, there have been no reports presenting evidence of transesterification between SCs and ethanol in vitro.Methods The in vitro metabolism of the four carboxylate SCs PB-22, NPB-22, 5-fluoro-PB-22 (5F-PB-22), and 5-fluoro-NPB-22 (5F-NPB-22) in the presence of ethanol using human liver microsomes with and without appropriate enzyme inhibi-tors was studied. Newly identified SC ethyl esters were chemically synthesised and fully characterised. The activity of these SCs and their ethanol transesterification products were assessed using cannabinoid receptor (CB1 and CB2) activation assays.Results SCs/ethanol transesterification products were detected and studied using liquid chromatography–high-resolution mass spectrometry. We have shown that the SC ethyl ester formation is mediated by human carboxyl esterase enzymes. The ethyl esters exhibited a reduced activity for the CB receptors compared with their parent compounds.Conclusions These novel ethyl esters may be useful additional markers of cannabinoid administration, and especially so if they prove to have longer half-lives than their parent compounds.

Keywords Carboxylate synthetic cannabinoids · In vitro drug metabolism · Transesterification · 5F-NPB-22 · Ethanol · Biomarkers

Introduction

The use of synthetic cannabinoid receptor agonists, or sim-ply ‘synthetic cannabinoids’ (SCs), is a global toxicologi-cal conundrum. Their use appears to have escalated during

2008–2015 [1] under the pretence that these compounds are ‘safe’ and ‘legal’ [2], and it is assumed that their use cannot easily be detected [3]. Although there has been a decline in seized SCs recently, many of the recent substances are highly potent and harmful [1]. Originally, these compounds, which circumvented drug legislation in many countries, were sold over-the-counter in so-called headshops and on the Internet as vividly branded products such as ‘Spice’ in

Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1141 9-019-00491 -0) contains supplementary material, which is available to authorized users.

* Vincenzo Abbate vincenzo.abbate@kcl.ac.uk

1 Department of Analytical, Environmental and Forensic Sciences, King’s Forensics, King’s College London, London SE1 9NH, UK

2 Department of Analytical, Environmental and Forensic Sciences, Drug Control Centre, King’s Forensics, King’s College London, London SE1 9NH, UK

3 Department of Drug Sciences, University of Catania, V.le A. Doria, 95125 Catania, Italy

4 Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium

5 Clinical Toxicology, Guy’s and St Thomas’ NHS Foundation Trust and King’s Health Partners, London, UK

6 Clinical Toxicology, Faculty of Life Sciences and Medicine, King’s College London, London, UK

7 Analytical Services International, St. George’s University of London, Cranmer Terrace, London SW17 0RE, UK

96 Forensic Toxicology (2020) 38:95–107

1 3

Europe and ‘K2’ in the United States. The weakness of the Misuse of Drugs Act [4] and incorporated schedules in the UK could not readily keep up with the rapidity of novel compounds reaching the market, but the more recent UK Psychoactive Substances Act (PSA) in 2016 [5] appears to be having an impact. While some data suggest that the use of novel psychoactive substances (NPS), including SCs, has decreased since the introduction of the PSA, other studies suggest little impact in the number of emergency department presentations, and their use remains prevalent and problem-atic [6]. As of the end of 2017, more than 670 NPS were monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), and 179 out of these were SCs [1]. In the UK, this is especially relevant in the prisoner population and amongst those facing homelessness [3, 7]. Not only are the clinical features of acute SC toxicity dif-ficult to identify and treat, particularly for the more recent third-generation SCs [6, 8, 9], but the analysis of these com-pounds in bulk-drug seizures and in biological samples can also be challenging, even using the most modern analytical instrumentation. Analysis is complicated by (1) the sheer number of SCs that exist [10, 11], (2) their physicochemical diversity, (3) the frequent formation of multiple (pharmaco-logically active) metabolites [10, 12–14], (4) their (in)stabil-ity and chemical transformations when ingested via smoking (i.e., their pyrolysis products [15–18]), (5) their potency, which transpires to low concentrations in biological samples, and (6) the limited availability of reference materials. To aid the identification of SCs and their metabolites, in vitro models based on incubation with human liver microsomes (HLMs) and other enzyme fractions [19], coupled with in silico predictions, are commonly used to determine the most likely metabolic routes of ‘new’ SCs [20]. In silico models are also extremely useful at predicting the pharmacology and potential toxicology of SCs [21, 22]. Such literature resources are invaluable for building large screening librar-ies to maximise the chances of detecting toxicologically relevant SC use in samples.

However, in substance-using populations, polydrug use is common [23]. Considerations of drug-drug interactions with SCs are important clinically [24], but possible or unexpected biotransformation due to co-ingested compounds should also be considered. For example, transesterification of cocaine following concomitant use of ethanol and cocaine is well known to produce the pharmacologically active metabolite cocaethylene [25, 26]. Similarly, transesterification has also been demonstrated for methylphenidate, resulting in the pro-duction of ethylphenidate (itself an NPS with misuse poten-tial) [27]. These reactions are primarily catalysed by human carboxylesterases (hCES) in the liver, which participate in phase I metabolism of xenobiotics. Transesterification of PB-22 can also be observed as a thermal degradation artefact when detecting by gas chromatography–mass spectrometry

(GC–MS) in the presence of methanol or ethanol as a sol-vent [28].

There is increasing evidence confirming the use of SCs together with ethanol. A recent United Nations Office on Drugs and Crime (UNODC) report [29] highlighted ethanol as the third most frequent substance found in SC-related fatalities. In addition, ester-based SCs including 5F-PB-22 were detected, together with ethanol and other substances in postmortem cases [30, 31]. ADB-CHMINACA (also known as MAB-CHMINACA) and ethanol were both detected in a patient admitted to hospit [32]. A further case reported a fatal poisoning with the synthetic cannabinoid AB-CHMI-NACA and ethanol [33]. Finally, blood and urine ethanol concentrations were reported in three fatalities related to the use of 5F-ADB, 5F-PB-22 and AB-CHMINACA (5F-PB-22 = 2.60 and 3.58 g/kg, AB-CHMINACA = 1.45 and 2.57 g/kg, 5F-ADB = 0.09 and 0.12 g/kg for blood and urine ethanol concentrations, respectively) [34]. It is apparent that the discovery of novel biomarkers of concomitant SC-etha-nol consumption should facilitate the interpretation of such analytical investigations and may allow both extending the detection time and assisting in the understanding of the pat-terns of toxicity associated with such polydrug abuse.

In this study, we report in vitro evidence from HLM incu-bation experiments for the transesterification of four ester-containing SCs, namely PB-22, NPB-22, 5F-PB-22, and 5F-NPB-22 (Fig. 1), in the presence of ethanol, giving rise to a further group of potential new indicators of combined SC/ethanol use. These compounds were chosen because of the presence of a labile ester bond in their structures, making them amenable to the described chemical reaction, as well as their prevalence [35].

To evaluate whether the transesterification products might be more biologically active than the parent compounds, we used an activity-based cannabinoid receptor assay [36–38] to investigate the in vitro receptor activity of the ethyl esters formed by transesterification of ethanol with these SCs.

Materials and methods

Chemicals and reagents

PB-22, NPB-22, 5F-PB-22 and 5F-NPB-22 were kindly pro-vided by TICTAC Communications (London, UK). Their identity and purity were confirmed by nuclear magnetic resonance (NMR) spectroscopy and high-performance liq-uid chromatography–ultraviolet spectroscopy (HPLC–UV) (% purity > 90%). Methanol, acetonitrile, formic acid (all LC–MS grade) were purchased from Fisher Scientific (Loughborough, UK). Trifluoroacetic acid (TFA), chloro-form-d (CDCl3, isotopic purity 99.8 atom %D) were from Sigma-Aldrich (Dorset, UK). Deionised water was purified

97Forensic Toxicology (2020) 38:95–107

1 3

to 18.2 MΩ cm resistivity using a PURELAB Ultra sys-tem (ELGA, Woodridge, UK). Aqueous phosphate buffer (0.5 mol/L, pH 7.4), pooled HLMs (150 donors), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system solutions A and B were pur-chased from Corning (Woburn, MA, USA); ethyl indazole-3-carboxylate and 1-bromo-5-fluoropentane from Fluoro-Chem (Hadfield, UK); 1,5-diiodopentane from Alfa Aesar (Lancashire, UK); ethyl indole-3-carboxylate and potas-sium tert-butoxide (tBuOK) from Aldrich (Dorset, UK); potassium hydroxide (KOH), bis-p-nitrophenylphosphate (BNPP), tetrahydrofuran (THF), hexane, ethyl acetate and ethanol from Sigma-Aldrich (St. Louis, MO, USA); anhy-drous THF from Acros Organics (Geel, Belgium).

Synthesis and characterisation of ethyl esters

Synthesis of the ethyl esters (compounds 10, 11, 14 and 15, Fig. 1) was carried out via nucleophilic substitutions of the N1 nitrogen atoms of (1) ethyl indazole-3-carboxylate and (2) ethyl indole-3-carboxylate, each with 1,5-diiodo-pentane and 1-bromo-5-fluoropentane, to form the non-fluorinated and fluorinated analytes, respectively. Ester

hydrolysis products (compounds 12, 13, 16 and 17, Fig. 1) were synthesised by alkaline hydrolysis of ethyl esters. Summary experimental data are given in supplementary material section S1. Ethyl esters were characterised using NMR (1H-NMR, 13C-NMR, homonuclear correlation spectroscopy—COSY, and heteronuclear single-quantum correlation spectroscopy—HSQC) (NMR, Bruker Avance DRX 400 MHz; Bruker BioSpin, Rheinstetten, Germany).

Further analysis was carried out using reversed-phase high-performance liquid chromatography with diode-array detection (HPLC–DAD, HP-1050) (Agilent Technologies, Santa Clara, CA, USA) using a 100 × 2.1 mm i.d. C8 column (5 µm, Kinetex®; Phenomenex, Torrance, CA, USA). Solu-tions of each compound [100 μg/mL in 0.1% (v/v) TFA in water] were eluted using a water/acetonitrile gradient (both containing 0.1% (v/v) TFA, ambient temperature, 31 min analysis time). Finally, solutions of each compound [300 ng/mL in 10% (v/v) acetonitrile in deionised water containing 0.3% (v/v) formic acid] were analysed by liquid chromatog-raphy–high-resolution mass spectrometry (LC–HRMS) to generate reference retention times and product ion (MS2) spectra for the in vitro incubation experiments.

Fig. 1 Chemical structures of 5F-NPB-22, NPB-22, PB-22 and 5F-PB-22 (top panel), their corresponding ethyl esters (mid-dle panel) and their hydrolysis product (bottom panel). SCs synthetic cannabinoids

98 Forensic Toxicology (2020) 38:95–107

1 3

In vitro incubation

The HLM method was based on that previously described [19]. Briefly, solutions containing 10 µg of each substrate (1 mg/mL, 10 µL) were added to Eppendorf LoBind™ tubes (Stevenage, UK), and evaporated to dryness. Buffer solution (0.1 mol/L aqueous phosphate buffer, pH 7.4, 500 µL), NADPH-regenerating solutions [50 µL solution A: 26 mmol/L NADP+, 66 mmol/L glucose-6-phosphate, and 66 mmol/L MgCl2 (aq); 10 µL solution B: 40 U/mL glucose-6-phosphate dehydrogenase in 5 mmol/L sodium citrate], and deionised water (390 µL) were added. The tubes were capped, vortex-mixed, and preincubated (37 °C) with continuous gentle shaking for 10 min using an Eppendorf ThermoMixer® (Stevenage, UK). Reactions were initiated by the addition of HLM solution [protein content 20 mg/mL in 250 mmol/L sucrose (aq), 50 µL], gently mixed by hand and were incubated using an Eppendorf ThermoMixer® (2 h, total reaction volume 1000 µL). A negative control was pre-pared by incubating each of the compounds with buffer solu-tion and water, omitting the HLM and NADPH solutions. For incubations with ethanol, the initial volume of deionised water added to the incubation mixture was reduced to 340 µL, and aqueous ethanol solution [5.86% (v/v), 50 µL] was added. The final concentration of ethanol was 50 mmol/L (0.23 g/dL). In addition, incubations were carried out in the presence of specific carboxylesterase (CES), and nonspe-cific esterase inhibitors (BNPP and saturated NaF solution, respectively). For both experiments, the initial volume of deionised water added to the incubation mixture was reduced to 290 µL, and aqueous ethanol solution [5.86% (v/v), 50 µL] and inhibitor solution [either 1 mmol/L BNPP, or 10% (w/v) NaF in water, 50 μL] was added. The final concentration of ethanol was maintained at 50 mmol/L.

After incubation, all reactions were quenched by the addition of ice-cold acetonitrile (250 μL). Quenched solu-tions were vortex-mixed and centrifuged (13,000 g, 15 min). Supernatants were transferred to glass autosampler vials for analysis by LC–HRMS (5 μL injection volume).

LC–HRMS

An UltiMate 3000™ HPLC system coupled to a Q Exactive™ high-resolution mass spectrometer (both Thermo Scientific, San Jose, CA, USA) was used. Xcalibur™ soft-ware (version 14.0) was used for instrument control and data analysis. Chromatographic separation was performed using an Acquity™ BEH C18 column (50 × 2.1 mm i.d., 1.7 μm) (Waters, Borehamwood, UK). Eluents consisted of 0.3% (v/v) formic acid in deionised water (eluent A) and in acetonitrile (eluent B). Column temperature was main-tained at 40 °C. The elution gradient started at 10% eluent B (2 min), ramped to 95% (over 5 min), and then decreased

immediately to 10% (over 1 min) prior to re-equilibration. The total analysis time was 10 min.

Heated electrospray ionisation (HESI-II) settings were: sheath gas flow-rate 70; auxiliary gas flow-rate 10; spray voltage 3.75 kV; capillary temperature 320 °C; S-lens radio frequency level 55.0; auxiliary gas temperature 350 °C. Full-scan acquisition parameters were: positive ionisation mode; scan range m/z 100–1000; resolution 70,000 full width at half maximum (FWHM); automatic gain control (AGC) tar-get 1 × 106. MS2 acquisition parameters were: parallel reac-tion monitoring mode (based on an inclusion list containing the appropriate precursor ions); scan range m/z 100–750; resolution 35,000 FWHM; AGC target 1 × 106. The isola-tion width was 1.2 amu. The normalised collision energy (NCE) was optimised manually using available standards and in-house synthesised products. For data review, total ion chromatograms were extracted using a ± 2.5 ppm extraction window based on the exact m/z values.

Cannabinoid receptor assay

To assess the biological activities of the compounds, live cell-based reporter assays that monitor protein-protein inter-actions via the NanoLuc Binary Technology were used. Here, the receptor activation is evaluated via the interac-tion between β-arrestin 2 (βarr2), a cytosolic protein, and the cannabinoid receptor CB1 or CB2. Both βarr2 and CB1/CB2 are fused to an inactive part of nanoluciferase. When CB1 or CB2 are activated by a ligand, βarr2 is recruited to the receptor, allowing interaction of the complementary nanoluciferase subunits, yielding a functional enzyme that generates a bioluminescent signal in the presence of the sub-strate furimazine. Details regarding the development of the stable CB1 and CB2 cell lines used here have been reported elsewhere [36]. The original human embryonic kidney (HEK) T293 cell line was provided by Prof. O. De Wever (Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Belgium).

The cells were routinely maintained at 37 °C, 5% CO2, under humidified atmosphere in Dulbecco’s modified Eagle’s medium (GlutaMAX™; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, 100 mg/L strep-tomycin and 0.25 mg/L amphotericin B. Stability of the cell lines was followed by flow cytometric analysis. For experi-ments, cells were deposited on poly-d-lysine coated 96-well plates (Thermo Fisher Scientific) at 5 × 104 cells/well and incubated overnight. The cells were washed twice with Opti-MEM® I Reduced Serum Medium (Thermo Fisher Scien-tific) to remove any remaining FBS, and 100 μL Opti-MEM® I was added. The Nano-Glo Live Cell reagent (Promega, Madison, WI, USA), a nonlytic detection reagent containing

99Forensic Toxicology (2020) 38:95–107

1 3

the cell permeable furimazine substrate, was prepared by 20-fold dilution of the Nano-Glo Live Cell substrate using Nano-Glo LCS Dilution buffer, and 25 μL were added to each well. Subsequently, the plate was placed into a TriStar2 LB 942 multimode microplate reader (Berthold Technolo-gies GmbH & Co., Bad Wildbad, Germany). Luminescence was monitored during the equilibration period until the sig-nal stabilised (15 min). We added 10 μL per well of test compounds, present as concentrated (13.5-fold) stock solu-tions in 50% (v/v) methanol in Opti-MEM® I. The lumi-nescence was continuously detected for 120 min. Solvent controls were analysed with all experiments. The final con-centration of methanol (3.7% v/v) has previously been veri-fied to be well-tolerated by the cells [36].

Curve fitting and statistical analyses were performed using GraphPad Prism software (San Diego, CA, USA). The results are represented as mean area under the curve (AUC) ± standard deviation (SD) with at least five replicates for each data point (obtained in three independent experi-ments). All results were normalised to the Emax of JWH-018 (= 100%), our reference compound. Curve fitting of concen-tration-effect curves via nonlinear regression (four param-eters logistic fit) was employed to determine EC50 (a meas-ure of potency) and the Emax (a measure of efficacy). The maximal effect of all compounds was determined by either the Emax (for the parent compounds) or the maximal effect that was achieved for the compound at high concentrations.

Results and discussion

Characterisation of compounds

Data from the characterisation experiments (NMR and HPLC–UV) for the synthesised compounds (10-17) are presented in supplementary material section S1. Reten-tion times, precursor ion m/z values (all [M+H]+), and major product ion m/z values for the parent compounds and the eight synthesised compounds generated using the LC–HRMS method are summarised in Table 1.

For the indoles PB-22 and 5F-PB-22, the characteristic major product ions observed were in agreement with previ-ously published data [39], resulting from successive losses of the hydroxyquinolinyl substructures (m/z 214.1228 and 232.1121 for PB-22 and 5F-PB-22, respectively) and the (fluoro-) alkyl chains (m/z 144.0445 and 144.0435 for both compounds).

Product ions spectra for the two indazoles, NPB-22 and 5F-NPB-22, showed analogous product ions (m/z 215.1183 and 233.1074 for NPB-22 and 5F-NPB-22, respectively, plus 145.0398 and 145.0389 for both compounds). In addition, the indazoles gave rise to fragment ions corre-sponding to losses of the quinolinyl substructures, i.e., of the same m/z as the precursor ions for compounds 13 and 12, the ester hydrolysis products (m/z 233.1288 and

Table 1 Summary LC–HRMS data for parent and synthesised compounds PB-22, NPB-22, 5F-PB-22, 5F-NPB-22, and metabolites 10-17

LC–HRMS liquid chromatography–high-resolution mass spectrometry, RT retention time

Compound (formula) RT (min) Precursor ion accurate m/z (Δ ppm)

Major product ion(s) accurate m/z (Δ ppm)

PB-22 (C23H22N2O2) 6.21 359.1759 (1.39) 214.1228 (0.93), 144.0445 (0.69)NPB-22 (C22H21N3O2) 6.29 360.1714 (1.94) 233.1288 (1.29), 215.1183 (1.86), 145.0398 (1.38)5F-PB-22 (C23H21N2O2F) 6.05 377.1652 (−2.12) 232.1121 (−4.74), 144.0435 (−6.25)5F-NPB-22 (C22H20N3O2F) 6.03 378.1603 (−2.38) 251.1178 (−4.78), 233.1074 (−4.72), 213.1015 (−3.28),

145.0389 (−4.83)PB-22 ethyl ester (15) (C16H21NO2) 6.52 260.1651 (2.31) 232.1336 (1.72), 214.1231 (2.34), 188.1439 (2.66),

132.0811 (2.27), 118.0654 (2.54)NPB-22 ethyl ester (11) (C15H20N2O2) 6.34 261.1598 (0.00) 233.1282 (−1.29), 215.1178 (−0.46), 145.0395 (−0.70)5F-PB-22 ethyl ester (14) (C16H20NO2F) 5.98 278.1556 (1.80) 250.1241 (1.20), 232.1136 (1.72), 206.1345 (2.43),

132.0811 (2.27)5F-NPB-22 ethyl ester (10) (C15H19N2O2F) 5.82 279.1503 (0.00) 251.1188 (−0.80), 233.1083 (−0.86), 213.1021 (−0.47),

145.0396 (0.00)PB-22 ester hydrolysis product (17) (C14H17NO2) 5.61 232.1334 (0.86) 214.1225 (−0.47), 188.1433 (−0.53), 132.0807 (−0.76),

118.0651 (0.00)NPB-22 ester hydrolysis product (13) (C13H16N2O2) 5.21 233.1291 (2.57) 215.1183 (1.86), 163.0505 (1.84), 145.0399 (2.07)5F-PB-22 ester hydrolysis product (16) (C14H16NO2F) 5.03 250.1244 (2.40) 232.1135 (1.29), 206.1344 (1.94), 132.0811 (2.27)5F-NPB-22 ester hydrolysis product (12)

(C13H15N2O2F)4.85 251.1192 (2.80) 233.1084 (−0.43), 213.1022 (0.00), 145.0397 (0.69)

5F-NPB-22 methyl ester (C14H17N2O2F) 5.48 265.1354 (2.64) 251.1195 (1.99), 233.1089 (1.72), 213.1029 (3.28)

100 Forensic Toxicology (2020) 38:95–107

1 3

251.1178 for NPB-22 and 5F-NPB-22, respectively). For 5F-NPB-22, a defluorinated product ion was also observed (m/z 213.1015) which was not observed in the case of 5F-PB-22.

For the ethyl ester and ester hydrolysis products of PB-22 (compounds 15 and 17), the major protonated car-bonyl-indole core fragment ion (m/z 144.0444) was not observed at any significant relative abundance. Instead, the proposed alkyl-indole and alkyl-indole carboxylic acid product ions (m/z 188.1439 and 232.1336, respectively) were the major product ions for PB-22 ethyl ester, com-pound 15 (Fig. 2), in addition to a small amount of the ion at m/z 214.1231 also observed for both compounds 15 and 17. The ethyl ester of PB-22 gave rise to a product ion at m/z 132.0811 and 118.0654, which were observed as major product ions of PB-22 ester hydrolysis, compound 17.

With respect to the ethyl esters and hydrolysis products of 5F-PB-22 (compounds 14 and 16), similar fragments as PB-22 ethyl ester and ester hydrolysis were observed. The major product ions were the alkyl-indole, m/z 206.1337; and product ions at m/z 250.1234, 232.1129 and 118.0650 being the fragments of alkyl-indole carboxylic acid, alkyl-indole carbaldehyde and indole group, respectively. The ethyl ester of 5F-PB-22 also gave rise to a product ion at m/z 132.0806.

Observed product ions of NPB-22 ethyl ester and 5F-NPB-22 ethyl ester included the alkyl-indazole carbox-ylic acid, alkyl-indazole carbaldehyde, and indazole car-baldehyde (m/z 233.1276, 215.1171, and 145.0391 for NPB-22, m/z 251.1187, 233.1082, and 145.0395 for 5F-NPB-22, respectively). The 5F-NPB-22 ethyl ester also yielded a defluorinated fragment (m/z 213.1020) (Fig. 3) as observed in MS2 result of 5F-NPB-22 parent compound. The ester

hydrolysis product ions of NPB-22 and 5F-NPB-22 gave similar results to those of their corresponding ethyl esters.

HLM incubation experiments

PB‑22 and 5F‑PB‑22

In the absence of ethanol, metabolism of PB-22 proceeded as previously described to produce a series of phase I metabolites [39]. The major metabolite (by relative abun-dance) was the ester hydrolysis product (compound 17). Of note, this compound was also observed at a lower abundance (approximately 1% of the parent compound) in the negative control samples, in which no HLMs were included in the incubation (see supplementary material S3.4). In addition to the ester hydrolysis product, a series of hydroxylated metabolites were observed, with charac-teristic MS2 fragments suggesting monohydroxylation on (1) the pentyl chain, (2) the indole core and (3) the quin-olinyl substructure, carbonylation of the pentyl chain, and successive hydroxylation products following ester hydroly-sis and N-dealkylation. When ethanol was present, in addi-tion to these metabolites, the previously synthesised and characterised PB-22 ethyl ester was also detected along with further peaks suggesting monohydroxylation of the ethyl ester (Fig. 4). No ethyl ester was observed when PB-22 was incubated with ethanol in the presence of either saturated NaF (nonspecific CES inhibitor) or 1 mmol/L BNPP (specific CES inhibitor) (see supplementary mate-rial S3.2-S3.3). The observed LC–HRMS analyses and the MS2 spectra of ethyl esters when SCs were incubated with HLM and ethanol matched with the synthetic standards (see supplementary material S2.1-2.8 and S1.28-1.35).

Fig. 2 Product ion (MS2) spec-trum (annotated with proposed fragment ion structures) for the ethyl ester of PB-22 (compound 15, exact m/z for protonated molecular ion 260.1645) with normalised collision energy (NCE) at 25

101Forensic Toxicology (2020) 38:95–107

1 3

A summary of the observed in vitro phase I metabolism, including transesterification in the presence of ethanol, for PB-22 is shown in Fig. 5.

For the fluorinated indole analogue 5F-PB-22, oxida-tive, reductive defluorination and oxidative defluorination to carboxylic acid products (see supplementary material

S3.8) were observed in addition to the metabolites seen for PB-22. In the presence of ethanol, and ethanol plus CES-inhibitor, similar results to its non-fluorinated analogue were observed.

These data demonstrate for the first-time evi-dence of hCES-mediated transesterification of selected

Fig. 3 MS2 spectrum (annotated with proposed fragment ion structures) for the ethyl ester of 5F-NPB-22 (compound 10, exact m/z for protonated molecular ion 279.1503) with NCE at 35

Fig. 4 Overlaid extracted ion chromatograms (exact mass ± 2.5 ppm) of the major detected PB-22 human liver microsome (HLM)-derived metabolites in the presence of ethanol: a = parent drug PB-22; b = monohydroxylation; c = carbonylation; d = monohydroxylation

with N-dealkylation; e = ester hydrolysis (17); f = ester hydrolysis with monohydroxylation; g = PB-22 ethyl ester (15); h = PB-22 ethyl ester with monohydroxylation

102 Forensic Toxicology (2020) 38:95–107

1 3

ester-containing SCs in the presence of ethanol, which gives rise to a new family of potential markers for SC and etha-nol concomitant use. In the case of PB-22 and 5F-PB-22, our findings suggested that the ethyl ester formation was catalysed by hCES (most likely hCES-1 subtype as in the case of cocaine metabolism), because no ethyl ester forma-tion was observed in the absence of the HLMs, nor when CES activity was inhibited (by either nonspecific or specific inhibitors). Furthermore, multiple hydroxylated metabolites of the ethyl esters were also observed, which may constitute additional useful (likely urinary) biomarkers of combined SC/ethanol use in toxicological analyses. However, PB-22 contains a labile ester bond in the structure. Thus, when using GC–MS with an elevated temperature in the presence of ethanol as solvent, transesterification to produce the ethyl ester of PB-22 can be observed [28].

NPB‑22 and 5F‑NPB‑22

As for the two indazoles, NPB-22 produced similar in vitro metabolites to 5F-NPB-22. Hydrolysis of the ester linkage was the major metabolic pathway for both indazole ana-logues, NPB-22 and 5F-NPB-22. A series of monohydroxy-lated ester hydrolysis products were observed. In addition, ester hydrolysis followed by oxidative defluorination was detected for 5F-NPB-22. Extracted ion chromatograms for the metabolites of 5F-NPB-22 from in vitro HLM experi-ments are presented in Fig. 6 and the proposed metabolic pathway of 5F-NPB-22 (without ethanol added) is presented in Fig. 7. To the best of our knowledge, this is the first report of the in vitro metabolism of 5F-NPB22.

In the presence of ethanol, the ethyl esters of NPB-22 and 5F-NPB-22 were both detected. Interestingly, a small amount of the ethyl ester was observed (approximately 0.5% of the parent compound by abundance) in the pres-ence of only buffer and ethanol (i.e., without any HLMs, see supplementary material S3.11 and S3.14). Indazole SCs clearly display more reactivity towards nucleophilic agents than their indole counterparts. Indeed, the NPB-22 family was more rapidly prone to hydrolysis, methanolysis and, therefore, ethanolysis (Fig. 8), even in the absence of any biocatalyst.

Incubation of ester hydrolysis products with HLM

To further elucidate the mechanism of formation of the newly detected SC ethyl esters, hydrolysed SC esters (com-pounds 12, 13, 16 and 17) were separately incubated with HLMs to monitor any ethyl ester product formation. This only yielded hydroxylated metabolites (data not shown). Incubation with HLMs and ethanol did not result in the formation of ethyl esters (illustrated in supplementary material S4). We concluded that the investigated biocata-lysed reactions were highly dependent on the nature of the substrate: SC-ethanol transesterification occurred; how-ever, esterification of the hydrolysed esters with ethanol was not observed. Indeed, hCES is well known to accom-modate ester, thioester, carbamate, and amide bonds in its active site [40–42], suggesting that the hydrolysed esters, bearing a free carboxylic acid, are not good substrates for this enzyme family.

Fig. 5 Proposed metabolic pathway for PB-22, inclusive of ethyl ester formation in the presence of ethanol. (a–h: refer to Fig. 4)

103Forensic Toxicology (2020) 38:95–107

1 3

Biological evaluation of PB‑22, NPB‑22, 5F‑PB‑22, 5F‑NPB‑22, their ethyl esters and their ester hydrolysis products

All parent compounds (PB-22, NPB-22, 5F-PB-22,

5F-NPB-22) showed a concentration dependent response in both the CB1 and CB2 receptor activation assays (Fig. 9). The EC50 and Emax values were determined as a measure of relative potency and efficacy, respectively (see supplemen-tary material S5.1). Both the EC50 and Emax values of PB-22

Fig. 6 Overlaid extracted ion chromatograms (exact mass ± 2.5 ppm) of 5F-NPB-22 HLM-derived metabolites (without ethanol): a = parent drug 5F-NPB-22; b = ester hydrolysis (12); c = ester hydrolysis with monohydroxylation; d = ester hydrolysis with oxidative defluorination

Fig. 7 Proposed metabolic pathways for 5F-NPB-22 (a–d refer to Fig. 6)

104 Forensic Toxicology (2020) 38:95–107

1 3

and 5F-PB-22 were in good correspondence with the previ-ously reported result using the same reporter assays [43].

The ester hydrolysis products and ethyl esters were also evaluated at both CB receptors (except the 5F-PB-22 hydrolysis product as this was not available for analysis,

but has previously been reported to have no activity at CB1 and CB2 [43]). At CB1 receptors, hydrolysis resulted in a strong reduction of the activity (to < 1% that of JWH-018) (Fig. 10a, exact values in supplementary material S5.2). The ethyl esters of PB-22 and 5F-PB-22 also showed a strongly

Fig. 8 Overlaid extracted ion chromatograms (exact mass ± 2.5 ppm) of 5F-NPB-22 in the presence of methanol and ethanol (without HLM); a = 5F-NPB-22; b = hydrolysis of 5F NPB-22; c = methanolysis of 5F-NPB-22; d = ethanolysis of 5F-NPB-22

Fig. 9 Concentration-dependent response at CB1 (a) and CB2 (b) upon stimulation with different SCs. Data are given as mean receptor activa-tion (%) ± standard deviation (SD) (n = 5–8), normalised to the Emax of JWH-018

105Forensic Toxicology (2020) 38:95–107

1 3

reduced CB1 activation (3.2 and 4.2% for the PB-22 and 5F-PB-22 ethyl esters, respectively). No significant CB1 receptor activation was found for the indazole (NPB) deriv-atives. When compared with the Emax of the parent com-pounds, the relative activity of the ester hydrolysis products did not exceed 0.16%, while that of the ethyl esters did not exceed 1.1%.

With respect to CB2 activity, hydrolysis of the ester in the parent compound resulted in a strong reduction of the activ-ity: only 6% CB2 receptor activation for the ester hydrolysis product of PB-22 was found and an almost complete loss in CB2 receptor activation was found for the ester hydrolysis products of NPB-22 and 5F-NPB-22 (Fig. 10b, exact values in supplementary material S5.3). For PB-22, the ethyl esters resulted in a reduced CB2 activation compared to the par-ent compound, although still a 35.5% CB2 receptor activa-tion (relative to JWH-018) was found at high concentrations (1 µM). The ethyl ester of 5F-PB-22 resulted in a 31.7% CB2 activation. For the ethyl esters of the NPB-derivatives, 11.7 and 11% CB2 receptor activation was found. When

compared to the Emax of the parent compounds, the activity of the ester hydrolysis products did not exceed 4.73%, while that of the ethyl esters did not exceed 28.6%. Because of the limited receptor activation at high concentrations, the contri-bution of these ethyl esters to the pharmacological profile of SCs is expected to be minimal. This is distinct from the CB1 and CB2 receptor activation by the hydroxyl-metabolites of (5F)-PB-22 (hydroxy-group on the pentyl side chain), where much higher activities (> 100% Emax JWH-018) were found [43].

Conclusions

Polydrug use is very common amongst illicit and recrea-tional drug users. Here, we have shown in vitro evidence of the formation of SC-ethyl esters through an hCES-catalysed transesterification. Given that there are a number of reports which describe fatal and nonfatal cases in which toxico-logical findings are indicative of combined use of SCs and

Fig. 10 The maximal effect obtained at CB1 (a) and CB2 (b). Bars assigned with an asterisk are not significantly different from basal levels. Data are given as the mean percent-age CB receptor activation (in comparison to the Emax of JWH-018) ± SD (n = 5–8). EtOH ethanol

106 Forensic Toxicology (2020) 38:95–107

1 3

ethanol, it is suggested that clinical and forensic laborato-ries should monitor the potential appearance of the reported ethyl esters and/or their hydroxylated metabolites to facili-tate interpretation of such cases. It is worth mentioning that detection of the described ethyl esters may be associated with several ester-containing SCs (common metabolites to a number of SCs); therefore, their detection should be accom-panied by the concomitant presence of parent drugs (often difficult to be detected in urine) or appropriate metabolites that are unique to a certain SC to avoid any ambiguous reporting. The elimination profiles are needed to understand their potential contribution to toxicity in humans using SCs, especially if they prove to have a longer half-life than the parent compound. However, the ethics of administration of these unlicensed substances to humans makes this difficult, and the relevance of animal studies is questionable.

Acknowledgements We are grateful to TICTAC Communications for the provision of SC standards. The authors acknowledge the Royal Thai Government Scholarship for supporting this work. Annelies Cannaert and Christophe Stove acknowledge the support from Ghent University-Special Research Fund (BOF; PDO026-18 and BOF project Grants No. 01N00814 and 01J15517).

Compliance with ethical standards

Conflict of interest Paul Dargan is a member of the Scientific Commit-tee of the European Monitoring Centre for Drugs and Drug Addiction and Chair of the Scientific Committee of the European Association of Poisons Control Centres and Clinical Toxicologists. All other authors have no conflict of interest to declare.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Open Access This article is distributed under the terms of the Crea-tive Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribu-tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

References

1. EMCDDA (2018) European drug report 2018: trends and develop-ments. Publications Office of the European Union, Luxembourg. https ://doi.org/10.2810/80033 1

2. Tai S, Fantegrossi WE (2017) Pharmacological and toxico-logical effects of synthetic cannabinoids and their metabo-lites. Curr Top Behav Neurosci 32:249–262. https ://doi.org/10.1007/7854_2016_60

3. Ralphs R, Williams L, Askew R, Norton A (2017) Adding spice to the porridge: the development of a synthetic cannabinoid mar-ket in an English prison. Int J Drug Policy 40:57–69. https ://doi.org/10.1016/j.drugp o.2016.10.003

4. Dargan PI, Hudson S, Ramsey J, Wood DM (2011) The impact of changes in UK classification of the synthetic cannabinoid receptor

agonists in ‘Spice’. Int J Drug Policy 22:274–277. https ://doi.org/10.1016/j.drugp o.2011.02.006

5. Smyth BP, Lyons S, Cullen W (2017) Decline in new psychoactive substance use disorders following legislation targeting headshops: evidence from national addiction treatment data. Drug Alcohol Rev 36:609–617. https ://doi.org/10.1111/dar.12527

6. Webb NE, Wood DM, Greene SL, Hunter LJ, Archer JRH, Dines AM, Dargan PI (2019) Change in the new psychoactive sub-stances associated with Emergency Department acute toxicity presentations associated with the introduction of the UK 2016 Psychoactive Substances Act. Clin Toxicol 57:36–41. https ://doi.org/10.1080/15563 650.2018.14942 77

7. Blackman S, Bradley R (2017) From niche to stigma—headshops to prison: exploring the rise and fall of synthetic cannabinoid use among young adults. Int J Drug Policy 40:70–77. https ://doi.org/10.1016/j.drugp o.2016.10.015

8. Waugh J, Najafi J, Hawkins L, Hill SL, Eddleston M, Vale JA, Thompson JP, Thomas SH (2016) Epidemiology and clinical features of toxicity following recreational use of synthetic can-nabinoid receptor agonists: a report from the United Kingdom National Poisons Information Service. Clin Toxicol 54:512–518. https ://doi.org/10.3109/15563 650.2016.11713 29

9. Cordeiro SK, Daro RC, Seung H, Klein-Schwartz W, Kim HK (2018) Evolution of clinical characteristics and outcomes of syn-thetic cannabinoid receptor agonist exposure in the United States: analysis of National Poison Data System data from 2010 to 2015. Addiction 113:1850–1861. https ://doi.org/10.1111/add.14281

10. Presley BC, Gurney SM, Scott KS, Kacinko SL, Logan BK (2016) Metabolism and toxicological analysis of synthetic cannabinoids in biological fluids and tissues. Forensic Sci Rev 28:103–169 (PMID:27257717)

11. Abbate V, Schwenk M, Presley BC, Uchiyama N (2018) The ongoing challenge of novel psychoactive drugs of abuse. Part I. Synthetic cannabinoids (IUPAC Technical Report). Pure Appl Chem 90:1255–1282. https ://doi.org/10.1515/pac-2017-0605

12. Brents LK, Reichard EE, Zimmerman SM, Moran JH, Fantegrossi WE, Prather PL (2011) Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity. PLoS One 6:e21917. https ://doi.org/10.1371/journ al.pone.00219 17

13. Chimalakonda KC, Seely KA, Bratton SM, Brents LK, Moran CL, Endres GW, James LP, Hollenberg PF, Prather PL, Radom-inska-Pandya A, Moran JH (2012) Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands. Drug Metab Dispos Biol Fate Chem 40:2174–2184. https ://doi.org/10.1124/dmd.112.04753 0

14. Schoeder CT, Hess C, Madea B, Meiler J, Müller CE (2018) Pharmacological evaluation of new constituents of “Spice”: syn-thetic cannabinoids based on indole, indazole, benzimidazole and carbazole scaffolds. Forensic Toxicol 36:385–403. https ://doi.org/10.1007/s1141 9-018-0415-z

15. Kaizaki-Mitsumoto A, Hataoka K, Funada M, Odanaka Y, Kuma-moto H, Numazawa S (2017) Pyrolysis of UR-144, a synthetic cannabinoid, augments an affinity to human CB1 receptor and cannabimimetic effects in mice. J Toxicol Sci 42:335–341. https ://doi.org/10.2131/jts.42.335

16. Raso S, Bell S (2017) Qualitative analysis and detection of the pyrolytic products of JWH-018 and 11 additional synthetic can-nabinoids in the presence of common herbal smoking substrates. J Anal Toxicol 41:551–558. https ://doi.org/10.1093/jat/bkx03 9

17. Thomas BF, Lefever TW, Cortes RA, Grabenauer M, Kovach AL, Cox AO, Patel PR, Pollard GT, Marusich JA, Kevin RC, Gamage TF, Wiley JL (2017) Thermolytic degradation of syn-thetic cannabinoids: chemical exposures and pharmacological

107Forensic Toxicology (2020) 38:95–107

1 3

consequences. J Pharmacol Exp Ther 361:162–171. https ://doi.org/10.1124/jpet.116.23871 7

18. Kevin RC, Kovach AL, Lefever TW, Gamage TF, Wiley JL, McGregor IS, Thomas BF (2019) Toxic by design? Formation of thermal degradants and cyanide from carboxamide-type syn-thetic cannabinoids CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA, MDMB-FUBINACA, NNEI, and MN-18 during exposure to high temperatures. Forensic Toxicol 37:17–26. https ://doi.org/10.1007/s1141 9-018-0430-0

19. Menzies EL, Hudson SC, Dargan PI, Parkin MC, Wood DM, Kicman AT (2014) Characterizing metabolites and potential metabolic pathways for the novel psychoactive substance meth-oxetamine. Drug Test Anal 6:506–551. https ://doi.org/10.1002/dta.1541

20. Diao X, Huestis MA (2017) Approaches, challenges, and advances in metabolism of new synthetic cannabinoids and identification of optimal urinary marker metabolites. Clin Pharmacol Ther 101:239–253. https ://doi.org/10.1002/cpt.534

21. Hess C, Schoeder CT, Pillaiyar T, Madea B, Müller CE (2016) Pharmacological evaluation of synthetic cannabinoids identified as constituents of spice. Forensic Toxicol 34:329–343. https ://doi.org/10.1007/s1141 9-016-0320-2

22. Floresta G, Apirakkan O, Rescifina A, Abbate V (2018) Dis-covery of high-affinity cannabinoid receptors ligands through a 3D-QSAR ushered by scaffold-hopping analysis. Molecules 23:e2183. https ://doi.org/10.3390/molec ules2 30921 83

23. Liu Y, Williamson V, Setlow B, Cottler LB, Knackstedt LA (2018) The importance of considering polysubstance use: lessons from cocaine research. Drug Alcohol Depend 192:16–28. https ://doi.org/10.1016/j.druga lcdep .2018.07.025

24. Kong TY, Kim JH, Kim DK, Lee HS (2018) Synthetic cannabi-noids are substrates and inhibitors of multiple drug-metabolizing enzymes. Arch Pharm Res 41:691–710. https ://doi.org/10.1007/s1227 2-018-1055-x

25. Jatlow P (1993) Cocaethylene: pharmacologic activity and clinical significance. Ther Drug Monit 15:533–536 (PMID:8122289)

26. Laizure SC, Mandrell T, Gades NM, Parker RB (2003) Cocaeth-ylene metabolism and interaction with cocaine and ethanol: role of carboxylesterases. Drug Metab Dispos 31:16–20. https ://doi.org/10.1124/dmd.31.1.16

27. Patrick KS, Corbin TR, Murphy CE (2014) Ethylphenidate as a selective dopaminergic agonist and methylphenidate-ethanol transesterification biomarker. J Pharm Sci 103:3834–3842. https ://doi.org/10.1002/jps.24202

28. Tsujikawa K, Yamamuro T, Kuwayama K, Kanamori T, Iwata YT, Inoue H (2014) Thermal degradation of a new synthetic cannabi-noid QUPIC during analysis by gas chromatography–mass spec-trometry. Forensic Toxicol 32:201–207. https ://doi.org/10.1007/s1141 9-013-0221-6

29. UNODC (2019) Current NPS threats, vol 1. UNODC Laboratory and Scientific Section, Vienna

30. Behonick G, Shanks KG, Firchau DJ, Mathur G, Lynch CF, Nashelsky M, Jaskierny DJ, Meroueh C (2014) Four postmor-tem case reports with quantitative detection of the synthetic cannabinoid, 5F-PB-22. J Anal Toxicol 38:559–562. https ://doi.org/10.1093/jat/bku04 8

31. Logan BK, Mohr ALA, Friscia M, Krotulski AJ, Papsun DM, Kacinko SL, Ropero-Miller JD, Huestis MA (2017) Reports of adverse events associated with use of novel psychoactive

substances, 2013–2016: a review. J Anal Toxicol 41:573–610. https ://doi.org/10.1093/jat/bkx03 1

32. Katz KD, Leonetti AL, Bailey BC, Surmaitis RM, Eustice ER, Kacinko S, Wheatley SM (2016) Case series of synthetic can-nabinoid intoxication from one toxicology center. West J Emerg Med 17:290–294. https ://doi.org/10.5811/westj em.2016.2.29519

33. Gieron J, Adamowicz P (2016) Fatal poisoning with the synthetic cannabinoid AB-CHMINACA and ethyl alcohol—a case study and literature review. Probl Forensic Sci 106:482–495

34. Angerer V, Jacobi S, Franz F, Auwärter V, Pietsch J (2017) Three fatalities associated with the synthetic cannabinoids 5F-ADB, 5F-PB-22, and AB-CHMINACA. Forensic Sci Int 281:e9–e15. https ://doi.org/10.1016/j.forsc iint.2017.10.042

35. Home Office (2018) Annual report on the Home Office Forensic Early Warning System (FEWS)–2015/16: a system to identify new psychoactive substances (NPS) in the UK. Information Policy Team, The National Archives, Kew, London

36. Cannaert A, Franz F, Auwärter V, Stove CP (2017) Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system. Anal Chem 89:9527–9536. https ://doi.org/10.1021/acs.analc hem.7b025 52

37. Cannaert A, Storme J, Hess C, Auwärter V, Wille SMR, Stove CP (2018) Activity-based detection of cannabinoids in serum and plasma samples. Clin Chem 64:918–926. https ://doi.org/10.1373/clinc hem.2017.28536 1

38. Cannaert A, Vandeputte M, Hudson S, Wood DM, Dargan PI, Stove CP (2019) Validation of activity-based screening for synthetic cannabinoid receptor agonists in a large set of serum samples. Clin Chem 65:347–349. https ://doi.org/10.1373/clinc hem.2018.29690 5

39. Wohlfarth A, Gandhi AS, Pang S, Zhu M, Scheidweiler KB, Huestis MA (2014) Metabolism of synthetic cannabinoids PB-22 and its 5-fluoro analog, 5F-PB-22, by human hepatocyte incuba-tion and high-resolution mass spectrometry. Anal Bioanal Chem 406:1763–1780. https ://doi.org/10.1007/s0021 6-014-7668-0

40. Wang D, Zou L, Jin Q, Hou J, Ge G, Yang L (2018) Human carboxylesterases: a comprehensive review. Acta Pharm Sin B 8:699–712. https ://doi.org/10.1016/j.apsb.2018.05.005

41. Laizure SC, Herring V, Hu Z, Witbrodt K, Parker RB (2013) The role of human carboxylesterases in drug metabolism: have we overlooked their importance? Pharmacotherapy 33:210–222. https ://doi.org/10.1002/phar.1194

42. Imai T, Taketani M, Shii M, Hosokawa M, Chiba K (2006) Sub-strate specificity of carboxylesterase isozymes and their contri-bution to hydrolase activity in human liver and small intestine. Drug Metab Dispos 34:1734–1741. https ://doi.org/10.1124/dmd.106.00938 1

43. Cannaert A, Storme J, Franz F, Auwärter V, Stove CP (2016) Detection and activity profiling of synthetic cannabinoids and their metabolites with a newly developed bioassay. Anal Chem 88:11476–11485. https ://doi.org/10.1021/acs.analc hem.6b026 00

Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.